Liu J L, Wang M K, Sun Q Y, Xu Z, Chen D Y
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Theriogenology. 2000 Oct 1;54(6):989-98. doi: 10.1016/S0093-691X(00)00407-6.
Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.
末期去核已被证明是在牛胚胎核移植中制备受体细胞质体的一种有效方法(2, 11)。本研究旨在探讨以处于末期II期、32小时龄的体外成熟去核卵母细胞作为受体细胞质体，与处于中期II期、24小时龄的卵母细胞相比，含有移植体细胞细胞核的牛卵母细胞的体外发育情况。采用了两种供体细胞注射方案，即透明带下注射(SUZI)和胞质内注射(ICI)。来自成年母牛的牛输卵管上皮细胞(BOECs)和牛卵丘细胞(BCCs)用作这些实验的核供体。在SUZI组中，BOECs和BCCs这两种供体细胞与MII期去核卵母细胞的融合率均高于与TII期去核卵母细胞的融合率(分别为54%对41%和53%对39%；P<0.05)，但MII组至桑葚胚加囊胚期的发育率低于TII组(分别为22%对39%和21%对41%；P<0.05)。在ICI组中，约26%注射了BOECs或BCCs的去核MII期卵母细胞发生了分裂，且只有一小部分发育到囊胚期(分别为4%和3%；P>0.05)。当将BOECs或BCCs胞质内注射到TII期去核的卵母细胞中时，两个供体细胞组均未形成囊胚，且BOEC组未发生分裂。我们的数据表明，当通过透明带下注射进行牛体细胞核移植时，末期去核有利于早期胚胎发育。然而，当将供体细胞直接注射到去核卵母细胞的细胞质体中时则是有害的。
